人类肝脏蛋白质组计划的实施与毒理学发展的机遇ppt课件.ppt

国际肝脏蛋白质组学研究计划的实施与毒理学发展机遇,1,报告提要,蛋白质组学产生的时代背景“人类蛋白质组计划”的目标与意义蛋白质组学与毒理学/环境医学,2,曼哈顿原子弹研制计划,人类基因组计划,阿波罗登月计划,人类历史上的三大科技工程,1941.12.61945.7.16罗斯福批准,耗资20亿美元,原子半径10-10m,原子体积10-30m3,人体半径100m,人体体积100m3,太阳系半径1012m,太阳系体积1034m3,1990.10.1-2003.4.23克林顿、布莱尔批准，耗资30亿美元,1961.5.251969.7.20肯尼迪批准,耗资240亿美元,3,人类基因组计划开创了“基因组时代”,4,基因组学向蛋白质组学“求助”!,Nature409:747,15Feb.2001Andnowforproteome“现在轮到蛋白质组”Science291:1221,16Feb.2001ProteomicsinGenomeland“基因组大地中的蛋白质组学”,5,PROTEIN,6,蛋白质组,一种细胞、组织或生物体所对应的全套蛋白质,PROTEIN,功能执行体,遗传信息载体,7,基因和蛋白质并不存在严格的线性关系ORF并不预示一定存在相对应的功能性基因mRNA水平并非与蛋白质的表达水平对应翻译后修饰及同工蛋白质(Isoforms)等现象在基因水平无从表现蛋白质与蛋白质的相互作用难以在基因水平得以认识,基因组与转录组不能取代蛋白质组,8,蛋白质调节的多样性,生命的“万花筒”,9,蛋白质功能的群体性,10,绳,墙,扇,茅,蛇,树,蛋白质作用的整体性,11,蛋白质组,生命体的统一性源于基因组生命体的多样性、复杂性、功能性、表型源于蛋白质组,1,46,3-4x104,3x109,103(1012),12,Pteomicsismoreofaconceptthanadefinedtechnology,anditreferstoanyprotein-basedapproachthathasthecapacitytoprovidenewinformationaboutproteinsonagenomewidescale.“Proteomicsincludesnotonlytheidentificationandquantificationofproteinsbutalsothedeterminationoftheir:localizationmodificationsinteractionsactivitiesfunction,13,报告提要,蛋白质组学产生的时代背景“国际人类肝脏蛋白质组计划”的目标与意义蛋白质组学与毒理学/环境医学,14,DNA序列图基因图人类基因组中1/3以上基因未曾确认基因确认的基本层次-蛋白质水平,天书,解读天书,Science291:1221,2001,15,蛋白质组,单基因病遗传病,多基因病肿瘤等,重大疾病发生发展机制,单一基因,单一蛋白,基因群,蛋白质群,转录组,蛋白质组,?,?,?,?,16,蛋白质组,6-30倍药靶有待发现,17,启动人类蛋白质组计划势在必行！,全面揭示重大疾病发生发展机制的基础,人类蛋白质组VIP:VeryImportantProteome,18,人类蛋白质组计划的主要科学目标,验证人类基因组计划推测的基因，注释基因组阐明蛋白质组的调控模式并与转录组进行对比建立蛋白质群/组装体，蛋白质复合物或蛋白质机器,即连锁图建立人体生理学、病理学的蛋白质组基础,19,1,46,3-4x104,3x109,103(1012),基因组计划,蛋白质组计划,20,21,Bodyfluidmostaccessibleforbiomarkerdiscoveryinanimalandhumans.,Liver,Kidney,Organ,BodyFluid,Brain,ImmuneOrgans/Vessels,Intestine,OtherTissues,CSF,Feces,Endocrine/ParacrineSecretions,Bile,Urine,Air/BALF,Lymph,Lungs,Eyes,Skin,OralCavity,Sweat,Saliva,Tears,VenousBlood,ArterialBlood,Serum,10%,90%,SerumProteins,Non-InformativeAbundantProteinsi.e.albumin,IgG,etc.,InformativeLowAbundanceProteins,EnrichmentStrategiesforInformativeSerumProteins:,SELDI,SerumImmunosubtraction,22,人类肝脏蛋白质组计划里程碑,蛋白表达谱蛋白连锁图亚细胞定位图修饰谱,人类基因组计划里程碑,遗传图物理图序列图,23,InitiativesofHumanProteomeProject(HPP),HPPPHumanPlasmaProteome,Project,USA,HLPPHumanLiverProteomeProject,China,PSIProteomicsStandardsInitiativeUK,HBPPHumanBrainProteomeProject,Germany,HAIHumanAntibodyInitiativeSweden,24,Whyisliver?,wwwww.HLPP.HUPO,为何研究肝脏?,25,肝脏功能的多样性与重要性,能量能量转换、储存物质代谢（活性分子的合成、毒性分子的分解）信息信息分子的合成与分泌,26,肝脏为其它组织提供能量可氧化底物,组织的活力依赖于高能键的连续生成。肝脏产生大部分脂肪酸，后者是禁食状态下能量的基本来源。肝脏是给食状态下由过剩糖合成脂肪酸的主要部位。,27,药物,毒物,营养物,生物异源物,生物转化,28,化学多样性向体内的生物转化体系提出严峻挑战,1.2107种以上的化学物(CAServiceslist)以每周约8000种的速率增加常用化学物在63,000种以上大约11,500种，作为食物或药物制剂添加物摄入其余的50,000种，为潜在的环境污染物,29,人类发育过程中造血组织的兴替,造血系统的发育必需肝脏的“培育”,30,合成大多数循环血浆蛋白,合成和分泌血浆蛋白是肝脏实质细胞肝细胞的独有功能（肝细胞占肝脏总细胞数的60%及肝脏质量的约80%）最有特征的血浆蛋白是白蛋白，在大多数哺乳动物中占总血浆蛋白的55-60%凝血酶、抗凝因子、溶栓酶等,31,肝脏再生,机体再生能力最强的器官终生保持旺盛的再生能力,32,肝脏中包含的“组”(-ome),MetabolicgenomicsMetabolome(代谢组)EnergygenomicsEnergome(能量组)PharmacogenomicsPharmacome(药理组)ToxicogenomicsToxicome(毒理组)RegeneratinggenomicsRegenerome(再生组),33,全球及中国肝炎的流行病学统计,全球:3.5亿携带者中国:1.8亿携带者死亡:23万人/年疾病负担:500亿元人民币治疗手段:抗病毒,保肝降酶,抗纤维化,免疫治疗等缺点:病毒易反弹,病情易反复,34,肝炎向肝癌的恶性转化难以遏制,全世界现有3.5亿慢性乙肝病毒（HBV）携带者，占世界人口的5%，亚洲和非洲HBV携带率为8-15%HBV携带者中50-70%病毒复制活跃，为慢性乙肝慢性乙肝病人肝硬化发生率为2-20%，代偿性肝硬化发展成失代偿性肝硬化为20-23%，发展成肝癌的占6-15%中国的慢性乙肝病人中，约25-40%最终将死于肝硬化或合并肝癌HBV携带者最终死于相关肝病的危险性，男性为50%，女性为15%,35,全球:100万人/年中国:50万人/年死亡:约45万人/年疾病负担:400亿元人民币治疗手段:手术切除(只有15%-20%)治疗效果:术后易转移,易复发,五年存活率40-50%,全球及中国肝癌的流行病学统计,36,原发性肝癌的治疗水平亟待突破,世界上每年有100万新发原发性肝癌病人，其中40-50%在我国我国原发性肝癌发病率和死亡率均位居第二位近20年来我国肝癌的发病率上升近40%原发性肝癌一般起病较隐匿，早期缺乏典型临床表现，初诊大多已属中晚期初诊肝癌病人中，只有15-20%的病人具备手术条件这些病人术后5年生存率仅为40-50%肝癌是致死率最高的恶性肿瘤之一,37,重大肝病的发生发展无法归咎于少数几个基因或蛋白质所呈现的功能状态和信号通路,从蛋白质组层面上“全景式”揭示肝脏疾病的生理病理机制是解决重大肝病的根本出路,肝炎病毒,肝脏/肝细胞,肝炎,肝硬化/纤维化,原发肝癌,肝癌转移,多因素、多步骤的发病机制,38,肝脏是人类蛋白质组计划的首批目标！,39,Prometheusstolefirefromthegodsandgaveittomortals.,PrometheusBound,HeraclesLiberatePrometheus,HLPPModernPrometheusMythPrometheusLiverEagleLiverdiseases,HeraclesHLPP,40,人类肝脏蛋白质组计划HumanLiverProteomeProject(HLPP),国际HLPP共同体,41,GenerateanintegrativeapproachleadingtoacomprehensivefunctionalmapoftheliverExpandliverproteometoits“PHYSIOME”and“PATHOME”todramaticallyacceleratethedevelopmentofdiagnostics,preventionandtherapeuticstowardsitsdiseasesDevelopstandardoperatingprocedures(SOPs)forotherHUPOInitiatives,VISION,42,HUPOWorkshop,Bethesda,April28-29,2002BroadinterestsandsupportsforinitiatingHLPPHUPOLiverProteomeWorkshop,Beijing,October22-24,2002GettingconsensusviewforscientificobjectivesofHLPPHUPO1stWorldCongress,Versailles,November21-24,2002Co-chairsofHLPP:Drs.FuchuHe,JohnBergeronandChristianBrchotHLPPPlanningCommittee:17members;May2003ProposalsaboutWorkingPlanofHLPPDr.JohnBergeron:Jan31,2003(Management);Dr.FuchuHe:Feb17,2003(ScientificStrategy)Dr.FuchuHe:Mar22,2003(ActionPlan);Dr.ChristianBrechot:Mar31,2003(Sampling)HLPPOffice,March18,2003HUPOLPPWorkshop,Bethesda,July17-18,2003NIHparticipatingHUPO2ndWorldCongress,Montreal,Oct.8-12,2003Dr.FuchuHewasappointedastheexecutiveChairofHLPP(2003-2005)HUPOHLPPSatelliteMeeting,Montreal,Oct.12,2003SetupExecutiveCommitteeDeltaCn0.1;RSp4.ABI-TOF/TOFProteinscore59(Rank1forthespot)Q-TOForQ-STARProteinscorethresholdPeptidescore“thescoreforidentity”or29MALDI-TOFProteinscorethresholdTheproteinsinfirstreport(multi-proteinforthemixture),59,Progress,EvaluationoftechnologiesBioinformaticsset-upSamplingandpreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissues,60,EvaluationoftheSOPsforthesubcellularfractionation,Object:FindtheoptimummethodofpretreatmentSample:LivertissuesofC57miceSubcellular:Mitochondria,Golgi,PM,Nucleus,ER,CytoplasmSamplepretreatmentFreshtissuesFrozenhomogenisedtissuesFrozentissues,61,Purity(Westernblotting):,Freshtissuesfrozenhomogenizedtissuesfrozentissues,Theyieldofproteins:,Freshtissuesfrozenhomogenizedtissuesfrozentissues,Integrity(TEM):,Freshtissuesfrozenhomogenizedtissues,Summary,62,Progress,EvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissues,63,Itisagiantambitiousobjectiveandgivesagreatchallengetoembryonicproteomics,Itisnecessarytocarryoutapreviewbeforetheformallaunchingofthisproject,Science302:1316,Nov.21,2003,Nature425:441,Oct.2,2003,64,AverageThroughputofthePlatform,2DESystem12Gel/3DaysAuto-SpotDigestSystem1152spots/DayMSandMS/MSSystem500-1000Spots/Day,65,TheflowchartofCCPITfortheproteinexpressionprofileofhumanfetalliver,66,Theareascalesbeforeandafterisoformprocessingingroupandproteinlevelsrespectively.Inproteinlevel,theredcircularityareasrepresentthenumberofconfirmedproteinsandtheyellowannulusareasrepresentthenumberofpossibleproteins.,f,o,r,m,p,o,s,s,i,b,l,e,:,1,2,5,8,67,ChromosomaldistributionofHFLproteome-encodinggenes,68,FunctionalModulesinplasmamembrane,69,Progress,EvaluationoftechnologiesBioinformaticsset-upSamplingandPreparationPreviewwithhumanfetalliverPrimaryanalysisofFrenchlivertissues,70,ReferenceLabsforHLPPExpressionProfilingProject,FuchuHe&XiaohongQian(BeijingInstituteofRadiationMedicine),RongZeng(ShanghaiInstituteforBiologicalSciences),PengyuanYang(FudanUniversity),SiqiLiu(BeijingGenomicsInstitute,ChineseAcademyofSciences),71,LauraBeretta(FHCRC,USA),AngelikaGorg(TechnischeUniversitatMunchen,Germany),MarkBaker/JunhongSun(APAF,Australia),72,Outline,BackgroundProgressoftheHLPPInitiationoftheCNHLPPNextwork,73,CNHLPPOverview,Thefirstphase:2004-2005Fundingfromcentralgovernment:10millionUSdollarsChineseMOST:jointlyfundedbyNationalProgramonKeyBasicResearchProjects(973),NationalHigh-techR&DProgram(863),andNationalKeyTechnologiesR&DProgrammeininthefirstphase.LocalandInstitutionalfunding:3.5millionUSdollars8subprojects70institutes,universities,andcompaniesinvolvedLong-termsupportasanationalproject(20062020),74,CelebrationofCNHLPPOfficialLaunching(BeijingInstituteofRadiationMedicine),75,Keylabs,BeijingInstituteofRadiationMedicine,76,Consultingcommittee(5),ORFeome&PPISubproject,ZeguangHan,ChairFuchuHe,HeadquartersBPRC,ModificationProfilesubproject,YunCai,ExpressionProfileSubproject,XiaohongQian,StructuralBiologySubproject,WeiminGong,Bioinformaticssubproject,YixueLi,NewTechnologysubproject,YukuiZhang,Antibodysubproject,Qi-HongSun,LocalizationMappingSubproject,XueminZhang,77,Morethan10workshopshavebeenheld,78,Briefprogressofsomesubprojects,ProteinmodificationProteinlocalizationProteinstructureNewproteomicstechnologies,79,AnalysisofproteinphosphorylationbycombinationofIMAC,PhosphatasewithBiologicalMassSpectrometry,m/z,Peptidemixture,Proteins,Candidatephosphopeptides,Identificationofphosphorylatedsites,DIGEST,IMAC,MALDI-TOFMSAnalysis,Phosphatasedigest,MS/MSAnalysisandDatabaseSearching,m/z,PhosphopeptideConfirmation,m/z,Metastableionsofphosphopeptides,80,IdentifiedphosphopeptidesequencesfromHFL,81,Proteinisolationandpurificationbylectin(Glycosylation),82,Co-localizationandlocomotionoftheGFPfusionproteinandDsRed,83,Structuralproteomics,Cloned900genesfromliverExpressedproductsofmorethan100genesDeterminedthestructuresof3proteinsbyX-rayDeterminedthestructuresof2proteinsbyNMR,84,Diphosphoglyceratemutase(DPGM),X-ray,Programmedcelldeath5(PDCD5),NMR,85,Beijing(National)ProteomeResearchCenter,ChineseProteomeDevelopmentCenter,86,TheAntibodyBankofHumanLiverProteomeProjectEstablishment,characterization,andapplicationofantibodiesagainsthumanliverproteinsQi-HongSunBeijingInstituteofRadiationMedicine(BIRM)BeijingProteomeResearchCenter(BPRC),87,Antigens,Myelomacells,Hybridomacells,mAbs,Diagnostics,Therapeutics,ProteomicsResearch,ProfilingIdentificationQuantificationLocalizationModificationInteractionFunction,Largeantibodycollectionforproteomics,88,AntibodyDatabase,AntibodyBank,Samples,5000liverproteins,HLPPAntibodyBank,Antibodychips,89,10%ofHLPPAntibodyBankStandardsanddatabaseofHUPOHLPP/HAIMicroarrayandotherantibody-basedtechnology,90%,10%,HLPPAntibodyBankatPilotPhase,90,AntigenpreparationFractionatedliverproteins-Unknown/native/multi-antigensRecombinantproteinsSynthesizedpeptidesAntibodygenerationHybridomacelllinesmAbs(murine/rabbit)PolyclonalantibodiesIgG(Rabbit)/orIgY(chicken)AntibodycharacterizationandapplicationClassandsubclass,ELISA,IH,WB,andIPAntibodymicroarrayandotherantibody-basedtechnology,GeneralApproach,91,IHcharacterizationofmAbsagainsthumanliverproteins,92,ProgresssummaryofHLPPAntibodyBankingProjectAntigenpreparationforimmunizationFractionatedliverproteinsnucleus,membrane,microsome,mitochondrial,cytosolic,andplasmaproteinsPurifiedrecombinantproteinantigens215Synthesizedpeptidesfromliver-specificproteins44AntibodypreparationandcharacterizationEstablishedhybridomacelllines1085CharacterizedmAbs862formorethan100differentproteinsRabbitpAbs50AntibodyApplicationDepletionofhigh-abundantproteinsImmunohistochemistryIsolationofproteincomplexes,93,LabsofHLPPAntibodyBankingProjectNational(China):Qi-HongSun/JianenGao,MingLi,MinZhao,DalingZhu,ChaonengJi,GangCheng,ZhinanChen,BoquanJing,JianWang/LinWu,XiaoningWang/YingLin,ZhengLi,JinliangYang,andJieTang.Internationalcollaboration:HUPO-HAI(Dr.Uhlen)FHCRC/NCI-IBDC(Dr.Lee)GermanSystemsBiologyProject(Drs.Meyer,Ueffing),94,WorkingplanAntibodydatasheetElectronicsubmissionformAntibodylistWebsite/HLPPAntibodydatabaseAntibodydemands/applicationsAntibodyqualitycontrolAntibodyexchange,collection,anddistributionScaleofantibodybankAntibody-basedtechnologyInternationalcollaboration,95,96,Outline,BackgroundProgressoftheHLPPInitiationoftheCNHLPPNextwork,97,Researchprojects,DistributetheChinesespecimenstoreferencelabsoutsideChinaEstablishtheSOPsfortheblackracialpeoplesliversampleComprehensivelyanalyzethedataoftheproteinsexpressingprofileoftheFrenchspecimensEstablishtheSOPsfortheprotein-proteininteractionsandORFeomeInitiatevirtuallydiseasedliverproteomesubprojectInitiatevirtuallyproteomicmodificationandlocalizationsubprojects,98,Coordination,Continuetodiscussthecooperationwithotherinitiatives(e.g.MRPP,HPPP,PSI,HAI,etc.)orotherprogramme(e.g.SystemsBiologyProjectofGermany)EstablishtheheadquartersoftheHLPPContinuetopropagandizetheHLPPtothepublicandprivatedomainsStriveformorefinancialsupportsfrommoreresources,99,HLPPworkshops,Jun.3,WashingtonOrganizer:LauraBeretaJun.10,EBI/LondonOrganizer:RolfApweilerAug.27,MunichOrganizer:FuchuHe,100,报告提要,蛋白质组学产生的时代背景“人类蛋白质组计划”的目标与意义蛋白质组学与毒理学/环境医学,101,主要作用,揭示环境因素的致病机制了解化学毒物的代谢途径解析微生物蛋白质组组成增强疾病预防诊断与治疗,102,GeneticsInfluenceEnvironment,Genomics,Transcriptomics,2-DE/MS,ICAT-LC/MS,InformationFlow,DNA,m-RNA,Protein,Proteinspecies2,Proteinspecies2,Proteinspeciesn,Ribosome,PTMs,Proteinspecies1,Proteinspecies1,Proteinspecies2,Proteomics,103,揭示环境因素的致病机制,蛋白质组学系统研究机体的蛋白质变化揭示环境因素对人体的致病机制,生物因素,化学因素,物理因素,104,ThemissionoftheintramuralProteomicsprogramwithintheNationalCenterforToxicogenomics(NCT)istoident